DNA sequence encoding the tumor suppressor gene ING1

ABSTRACT

The invention provides novel tumor suppressor genes, methods for making and using these and related tumor suppressor genes and proteins and peptides, and nucleic acids encoding these and related tumor suppressor proteins and peptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional, of Application Ser. No. 08/751,230 ,filed Nov. 15, 1996, which was a Continuation-In-Part of ApplicationSer. No. 08/569,721 filed Dec. 8, 1995.

FIELD OF THE INVENTION

This invention relates to a novel tumor suppressor gene, ING1, tomethods for making and using this and related tumor suppressor genes andproteins and peptides, and to nucleic acids encoding this and relatedtumor suppressor proteins and peptides.

REFERENCES

The following references are cited in the application as numbers inbrackets ([]) at the relevant portion of the application.

1. Levine, A. J., "The Tumor Suppressor Genes", Annu. Rev. Biochem. 62:623-651 (1993).

2. Hunter, T. et al., "Cyclins and Cancer II: Cyclin D and CDKInhibitors Come of Age", J. Cell 79: 573-582 (1994).

3. Gudkov, A. V. et al., "Isolation of genetic suppressor elements,inducing resistance to topoisomerase II-interactive cytotoxic drugs,from human topoisomerase II cDNA", Natl. Acad. Sc. USA 90: 3231-3235(1993).

4. Straus, D. et al., "Genomic subtraction for cloning DNA correspondingto deletion mutations", Proc. Nati. Acad. Sc. USA 87: 1889-1893 (1990).

5. Lisitsyn, N. et al., "Cloning the Differences Between Two ComplexGenomes", Science 259: 946-951 (1993).

6. Yaswen, P. et al., "Down-regulation of a calmodulin-related geneduring transformation of human mammary epithelial cells", Proc. Natl.Acad. Sc. USA 87: 7360-7364 (1990).

7. Miller, A. D. et al., "Improved Retroviral Vectors for Gene Transferand Expression", Biotechniques 7: 980-986 (1989).

8. Serrano, M. et al., "A new regulatory motif in cell-cycle controlcausing specific inhibition of cyclin D/CDK4", Nature 366: 704-707(1993).

9. Defeo-Jones, D., "° Cloning of cDNAs for cellular proteins that bindto the retinoblastoma gene product", Nature 352: 251-254 (1991).

10. Aharon, T. et al., "Selective Destabilization of Short-Lived mRNAswith the Granulocyte-Macrophage Colony-Stimulating Factor AU-Rich 3'Noncoding Region is Mediated by a Cotranslational Mechanism", Mol. Cell.Biol. 13: 1971-1980 (1993).

11. Guan, K. et al., "Growth suppression by p18, a p16^(INK4/MTS1) andp14^(INK4B/MTS2) -related CDK6 inhibitor, correlates with wild-type pRbfunction", Genes & Dev. 8: 2939-2952 (1994).

12. Harper, J. W. et al., "The p21 Cdk-Interacting Protein Cip1 is aPotent Inhibitor of G1 Cyclin-Dependent Kinases", Cell 75: 805-816(1993).

13. El-Deiry, W. S. et al., "WAF1, a Potential Mediator of p53 TumorSuppression", Cell 75: 817-825 (1993).

14. Kamb, A. et al., "A Cell Cycle Regulator Potentially Involved inGenesis of Many Tumor Types", Science 264: 436-440 (1994).

15. Nobori, T. et al., "Deletions of the cyclin-dependent kinase-4inhibitor gene in multiple human cancers", Nature 368: 753-756 (1994).

16. Riabowol, K. et al., "The cdc2 Kinase Is a Nuclear Protein That IsEssential for Mitosis in Mammalian Cells", Cell 57: 393-401 (1989).

17. Sambrook, J. et al., "Molecular Cloning" (2nd.Ed.), A LaboratoryManual, Cold Spring Harbor Laboratory Press (1989).

18. Harlow, E. et al., "Antibodies", A Laboratory Manual, Cold SpringHarbor Laboratory (1988) .

19. Yang, Y. et al., "An approach for treating the hepatobiliary diseaseof cystic fibrosis by somatic gene transfer" Proc. Nat'l. Acad. Sci. USA90: 4601-4605 (1993).

20. Atadja, P. et al., "Increased activity of p53 in senescingfibroblasts" Proc. Nat'l. Acad. Sci. USA 92: 8348-8352 (1995).

21. Demetrick, D. J. "Fluorescence in situ hybridization and human cellcycle genes" In the Cell Cycle--Materials and Methods M. Pagano (ed.)Springer Verlag Press, 29-45 (1995).

22. Motomura et al., "Loss of alleles at loci on chromosome 13 in humanprimary gastric cancers" Genomics 2, 180-184 (1988).

23. Mitelman et al., "Report of the committee on chromosome changes inneoplasia" Cytogenet Cell Genet 55: 358-386 (1990).

24. Maestro et al., "Chromosome 13q deletion mapping in head and necksquamous cell carcinomas: identification of two distinct regions ofpreferential loss" Cancer Research 56: 1146-1150 (1996).

25. Thompson, M. E. et al., "Decreased expression of BRCA-1 acceleratesgrowth and is often present during sporadic breast cancer progression"Nature Genetics 9: 444-450 (1995).

26. Pear, W. S. et al., "Production of high titer helper-freeretroviruses by transient transfection" Proc. Natl. Acad. Sci. 90:8392-8396 (1993).

27. Wong, H. et al., "Monitoring mRNA expression by polymerase chainreaction: the "primer-dropping" method" Anal. Biochem. 223: 251-258(1994).

28. Schneider E. L and Fowlkes, B. J., "Measurement of a DNA content andcell volume in senescent human fibroblasts utilizing flow miltiparametersingle cell analysis" Exp. Cell. Res. 98: 298-302 (1976).

29. Tsai, L. H. et al., "The cdk2 kinase is required for the G1- to -Stransition in mammalian cells" Oncogene 8: 1593-1602 (1993).

30. Bond, et al., "Escape from senescence in human diploid fibroblastsinduced directly by mutant p53" Oncogene 9: 1885-1889 (1994).

The disclosure of the above publications, patents and patentapplications are herein incorporated by reference in their entirety tothe same extent as if the language of each individual publication,patent and patent application were specifically and individuallyincluded herein.

BACKGROUND OF THE INVENTION

Many cancers originate and progress by is accumulating mutations in oneor more genes. Such mutations which result in cancer formation can be inproto-oncogenes or in tumor suppressor genes. Mutations in tumorsuppressor genes result in loss of function, and therefore act in arecessive fashion to native genes. Oncogenes, in contrast, act indominant fashion to native alleles and, therefore, are not usuallyinherited through the germ lines. The tumor suppressor genes, however,are found in inherited predispositions to cancer and are inherited as adominant predisposition because of the high frequency of a secondgenetic event such as reduction in homozygosity[1].

Several tumor suppressor genes have been identified. Examples includethe Rb gene, which is involved in retinoblastoma and osteosarcoma; p53,which is involved in osteosarcoma and adrenocortical, breast and braincancers; WT-1, which is involved in Wilms' tumor, nephroblastoma andneurofibromatosis; adenomatous polyposis coli (APC), which is involvedin adenomatous polyposis; and deleted colorectal cancer (DCC), which isinvolved with a somatic mutation in the colon.

The negative regulation of cell growth is effected by tumor suppressorproteins that regulate the cell cycle by different mechanisms [2]. Thegene cloned and sequenced as described herein, ING1 (formerly calledp33^(IG1)), represents a new tumor suppressor gene which is expressed innormal mammary epithelial cells, but expressed only at lower levels inseveral cancerous mammary epithelial cell lines and is not expressed inmany primary brain tumors.

Known applications of sequenced genes include use of the DNA sequence(or analogs thereof) or of RNA or amino acid sequences derived fromthese DNA sequences for diagnosis or treatment of the correspondingdisease. Accordingly, the gene ING1 (previously designated p33^(IG1)) isuseful for the diagnosis and treatment of breast and brain cancers amongothers.

SUMMARY OF THE INVENTION

The present invention is directed to a novel DNA sequence for anisolated gene (designated ING1). The DNA sequence, an RNA sequenceidentical to or complementary to the DNA sequence; the protein the DNAsequence encodes, p33^(ING1), and/or fragments or analogs thereof andantibodies which bind to p33^(ING1) which are useful for diagnosingand/or treating cancer.

One aspect provides a DNA sequence selected from the group consisting ofa DNA isolate substantially identical to the p33^(ING1) DNA sequenceshown in FIG. 2 and a DNA sequence greater than about 10 base pair (bp)in length capable of hybridizing under stringent conditions to thecomplement of the p33^(ING1) DNA sequence shown in FIG. 2. Recombinantexpression vectors comprising such DNA isolates and cells transformedwith such recombinant expression vectors are also provided.

Another aspect of the invention provides peptide or protein encoded bythe DNA sequence substantially identical to the DNA sequence of FIG. 2or a DNA sequence greater than about 10 base pair (bp) in length capableof hybridizing under stringent conditions to the complement of thep33^(ING1) DNA sequence shown in FIG. 2.

Another aspect of the invention provides a method for decreasingproliferation of mammalian cells comprising selecting said mammaliancells whose proliferation is to be decreased and increasing theexpression of p33^(ING1) in said mammalian cells. It is comtemplatedthat said mammalian cells may be selected from the group consisting ofnormal cells and cancerous cells. It is further contemplated that themethod of decreasing the expression of p33^(ING1) will compriseintroducing into said mammalian cells at least one composition selectedfrom the group consisting of p33^(ING1) and nucleotides which code forp33^(ING1).

Another aspect of the invention provides a method of diagnosing breastcancer comprising obtaining a biological sample comprising mammary cellssuspected of being neoplastic and determining whether or not thebiological sample contains p33^(ING1) or the DNA which encodesp33^(ING1), wherein the presence of p33^(ING1) or its DNA denotesnon-cancerous cells.

Another aspect of the invention provides a method of diagnosing breastcancer comprising obtaining a biological sample comprising mammary cellssuspected of being neoplastic, contacting said biological sample with atleast one antibody to p33^(ING1) under conditions wherein antibodybinding to p33^(ING1) occurs; and detecting whether or not said antibodybinds to said cells, wherein binding to said cells indicates that saidcells are non-cancerous.

One aspect of the invention provides nucleic acid isolates greater than10 nucleotides in length which are substantially identical to the DNAsequence of FIG. 3 or its complement. Recombinant expression vectorscomprising such sequences and cells transformed with such recombinantexpression vectors are also provided.

One other aspect of the invention provides for a nucleic acid sequencewhich encodes the amino acid sequence of FIGS. 2 or 3 and recombinantexpression vectors comprising such sequences and cells transformed withsuch recombinant expression vectors.

One other aspect of the invention provides for peptides and proteinshaving p33^(ING1) biological activity. It is contemplated that suchpeptides will have an amino acid sequence substantially identical to theamino acid sequence set forth in FIGS. 2 or 3.

Still a further aspect of the invention provides for antibodies to thep33^(ING1) protein.

A further aspect of the invention provides methods for decreasingproliferation of cancer cells in a patient comprising administering aneffective amount of the above-described nucleic acid isolates, nucleicacid sequences, proteins or peptides under conditions wherein p33^(ING1)is expressed in the cancer cells. It is contemplated that the cancer isselected from the group consisting of breast and brain cancers.

Another further aspect of the invention provides methods of increasingcell proliferation of mammalian cells by decreasing expression ofp33^(ING1) in the cells. It is contemplated that such methods couldinclude the administration of either a single-stranded oligonucleotidecomprising a sequence substantially identical to the complement of thecDNA sequence of FIG. 3 or the administration of a single ordouble-stranded oligonucleotide under conditions that a single-strandedoligonucleotide comprising a sequence substantially identical to thecomplement of the cDNA sequence of FIG. 3 is expressed in the cells. Itis further contemplated that chemical inhibitors of p33^(ING1) activitycould also be administered.

A yet further aspect of the invention provides a method for diagnosingcancer comprising obtaining a biological sample comprising cellssuspected of being neoplastic and detecting whether or not thebiological sample contains only the native ING1 gene, or expressesnative ING1 mRNA or p33^(ING1), wherein the presence of only the nativeING1 gene or expression of native ING1 mRNA or p33^(ING1) denotesnon-cancerous cells. Preferably the cancer is breast cancer or braincancer.

A still further aspect of the invention provides a kit for the detectionof neoplastic cells in a biological sample comprising cells suspected ofbeing neoplastic comprising a solid support for attaching the mRNA fromthe cell or tissue to be tested and a labelled polynucleotide of atleast 10 nucleotides which polynucleotide is substantially identical tothe sequence of FIG. 3 or its complement.

A still further aspect of the invention provides a kit for the detectionof neoplastic cells in a biological sample comprising cells suspected ofbeing neoplastic comprising a solid support for attaching the cells, ananti-p33^(ING1) antibody and a detectable label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a to 1c illustrate the strategy and biological assays used forcloning ING1.

FIG. 2 sets forth the partial cDNA sequence of ING1 (SEQ ID NO: 1) andthe predicted amino acid sequence (SEQ ID NO: 2) of p33^(ING1).

FIG. 3 sets forth the complete cDNA sequence of ING1 (SEQ ID NO: 9) andthe predicted amino acid sequence (SEQ ID NO: 10) of p33^(ING1).

FIGS. 4a to 4c illustrate the effects of p33^(ING1) overexpression.

FIG. 5 illustrates the changes in p33^(ING1) protein levels in breastcancer cell lines. FIG. 5a is a Western blot. FIG. 5b is a picture ofthe coomassie-blue stained gel of FIG. 5a.

FIG. 6a illustrates Western blotting of neuroblastoma cell lines withanti-p33^(ING1) antibody. FIG. 6b illustrates a Southern blot ofneuroblastoma cell lines for ING1 DNA. FIG. 6c illustrates the RT-PCRreaction on a neuroblastoma cell line compared to a control diploidfibroblast.

FIG. 7 illustrates the level of ING1 mRNA in control (c) tissue,glioblastoma (GB), astrocytoma (AS) and meningioma (MN) tumors asdetermined by RT-PCR.

FIGS. 8a and 8b illustrate the expression of ING1.mRNA and p33^(ING1) inproliferation competent (y) and in senescent human fibroblasts (o).

FIG. 9 illustrates the level of p33^(ING1) protein through the cellcycle. Panel A has anti-cdk2 antibody as a positive control. Panel Bshows the results with anti-p33 antibodies. Panel C shows cell cycleprofile at each point as determined by FACS.

FIG. 10 illustrates the number of cells per colony of cells blocked forING1 expression.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein relates to the discovery of a novel tumorsuppressor gene, designated ING1, expression of which is found in normalmammary epithelial cells and in normal brain cells but found only atlower levels in several breast cancer cell lines and which is absent ina majority of primary brain tumors including gliomas, meningiomas andastrocytomas.

Using a strategy based upon subtractive hybridization of normal andcancerous mammary epithelial cell mRNAs and the selection of geneticsuppressor elements [3], a novel gene was isolated encoding a 33 kDaprotein that is a potent inhibitor of cell growth. Acute expression oftransfected constructs encoding this gene inhibited cell growth asestimated by decreased S-phase fraction and blocked entry into S-phasefollowing needle microinjection. Chronic expression of antisenseconstructs resulted in tumor induction in vivo and in focus formation invitro, and also conferred the ability to grow in soft agar.

A. Definitions

As used herein the following terms have the following meanings:

"Antibody" means a molecule that binds to a known antigen. An"anti-p33^(ING1) antibody" means an antibody molecule that binds to oneor more epitopes of the p33^(ING1) protein.

"Antisense" and "Antisense nucleotides" means DNA or RNA constructswhich block the expression of the naturally-occurring gene product. Forexample, in the present invention, use of a DNA construct that producesING1 antisense RNA blocks the expression of p33^(ING1) by destroying orinactivating ING1 mRNA.

"Biological sample" means a sample of mammalian cells. These cells maybe part of a tissue or organ sample obtained, for example, by biopsy, orthey may be individual cells, for example, blood cells or cells grown intissue culture.

"Cancerous cell" means a cell in or from a neoplasm. Preferably thecancerous cells is breast cancer, brain cancer, gastric cancer,haematologic neoplasms and head and neck squamous cell carcinomas.

"Breast cancer" means any of various malignant neoplasms of the breastor mammary tissue.

"Brain cancer" means any of various malignant neoplasms of the brain,neuroglial cells or meninges.

"Cell cycle" means the cyclic biochemical and structural eventsoccurring during growth of cells. The cycle is divided into periodscalled : G_(o), Gap₁ (G₁), DNA synthesis (S), GAP₂ (G₂), and mitosis(M).

"Cell division" means mitosis, i.e., the usual process of cellreproduction.

"Cell-proliferation-inhibiting-peptide compound" means a peptide orpeptide-containing compound which inhibits cell proliferation, either invitro or in vivo.

"Code" or "encode", when used with reference to a nucleotide's relationto a protein, mean the system whereby particular combinations ofadjacent nucleotides control the insertion of particular amino acids inequivalent places in a protein molecule.

"Expression" means the production of a protein or nucleotide in thecell.

"Growth" means progression through the cell cycle with the result thattwo daughter cells are formed from each mother cell. "Actively growing"means that state wherein cells exhibit growth and cell division.

"Hyperplasticity" means an increase in cell number, excluding tumorformation.

"Label" means to incorporate into a compound a substance that is readilydetected. Such substances include radioactive substances and fluorescentdyes, for example.

"Mammalian cell" means a cell in or from a mammal, either in a tissue ororgan or in tissue culture.

"Neoplasia" means the process resulting in the formation and growth ofan abnormal tissue that grows by cellular proliferation more rapidlythan normal, and continues to grow after the stimuli that initiated thenew growth cease.

"Neoplastic" describes the abnormal tissue that grows by cellularproliferation more rapidly than normal, and continues to grow after thestimuli that initiated the new growth cease.

"Normal cell" means a non-cancerous cell.

"Proliferation" means growth and reproduction, i.e., division of cells.

"Native" means the nucleic acid of a non-mutated gene or peptidesequence encoded by such a gene as found in a phenotypically normalcell.

"Substantially identical" means that the polynucleotide or nucleic acidof interest is able to hybridize to the complement of the known sequenceunder stringent conditions. Such stringent conditions preferably requireat least 85% identity, more preferably the conditions require at least90% identity and most preferably the conditions require at least 95%identity. When used in relation to peptides and proteins, "substantiallyidentical" means that the amino acid sequence of the peptides share atleast 85% identity, more preferably at least 90% identity and mostpreferably at least 95% identity.

B. Synthesis and Methodology

To identify gene products whose inactivation might contribute to theemergence and growth of cancer cells, a novel positive selectionprocedure that combines subtractive hybridization with an in vivoselection assay was used to identify putative growth-suppressorelements. An overview of the strategy used is shown in FIG. 1a.

Following a modified subtractive hybridization protocol [4,5], totalcDNA from a normal mammary epithelial cell line [6] was hybridizedindependently with cDNAs from the breast cancer cell lines MCF-7,BT-483, BT-474, Hs-578T, ZR-75, MD-MB-468, MD-MB-435 and BT-20 whichwere obtained from the American Type Culture Collection. SubtractedcDNA, theoretically containing sequences more highly expressed in thephenotypically normal epithelial cells, was then used as a probe toscreen a normal human fibroblast cDNA library.

Following screening, 300 cDNA clones were isolated, and their insertswere digested into fragments of 200-800 base pairs. The fragments werethen recloned into the retroviral plasmid vector pLNCX [7]. Afterpassage through the packaging line BOSC 23 [3], retroviruses containingthe isolated fragments were used to infect normal mouse mammaryepithelial cells (NMuMG). The infected cells were subsequently injectedinto nude mice.

Within 45 days, several mice developed tumors from which the clonedinserts were recovered by amplification using primers specific for pLNCXin polymerase chain reactions (PCR). Two different sequences wereisolated from tumors, one of which was subsequently shown to beexpressed in the antisense orientation.

The antisense sequence isolated, when introduced into normal fibroblastcells, consistently showed the biological effects of increased cellproliferation in soft agar and in focus forming assays (FIG. 1b andTable 1) This 182 bp fragment represented nucleotides 781 to 963 of thecDNA shown in FIG. 2 and nucleotides 942 to 1124 of FIG. 3. This cDNAencodes a 33 kDa protein called p33^(ING1) for INhibitor of Growth. Thiswas formerly designated p33^(ING1) (see U.S. Ser. No. 08/569,721 whichis incorporated herein by reference in its entirety).

After plating NMuMG cells infected with either control virus or withvirus containing an insert of the antisense orientation of ING1 in softagar, cells receiving the insert formed, on average, at least 50 timesthe number of colonies as cells infected with virus alone. Similarresults were obtained following transfection of the retroviral constructinto NIH3T3 cells, where pLNCX containing the insert of the antisenseorientation of ING1 resulted in the formation of 2.3 times the number ofgenerally larger foci than vector alone.

These results corroborated the observations made in the nude mouse assaythat the ING1 sequence corresponds to a gene whose product plays asignificant role in regulating cell growth.

In order to isolate the gene corresponding to the fragment showingbiological effects, normal human fibroblast and HeLa cell libraries werescreened with the fragment, resulting in the isolation of 11 positiveclones. Two clones contained cDNA whose sequence is shown in FIG. 2. Thecomplete cDNA sequence (FIG. 3) was obtained using rapid amplificationof cDNA ends (RACE) by methods known in the art.

Comparison of the sequence of p33^(ING1) shown in FIG. 3 to theavailable protein and nucleotide data bases showed no significanthomology to any sequence encoding a known protein and very limitedsimilarity to retinoblastoma binding protein 2 (RbBP2) [9] and toseveral zinc finger transcription factors. Regions of the p33^(ING1)protein that show homology to retinoblastoma binding protein 2 wereidentified using the Blast program available from the National Centrefor Biological Information (address: www.ncbi.nim.nih.gov).

Use of a polyclonal antibody raised against a glutathione-S-transferase(GST) fusion with p33^(ING1) revealed a protein of 33 kDa by Westernblot analysis of human and mouse cell extracts (FIG. 1c)

To determine whether the level of p33^(ING1) was decreased in cellsinfected with viral constructs containing the antisense orientation,lysates were prepared from control NMuMG cells and from NMuMG cellsinfected with antisense ING1 that had grown and formed colonies insemi-solid medium. Results of Western blot analysis showed that chronicexpression of antisense construct reduced the expression of theendogenous p33^(ING1) protein by approximately 90% in the cells (FIG.1c, lane 6) compared to control parental cells (FIG. 1c, lane 5).

The ING1 CDNA contains several AU-rich elements (AREs) in the 3'untranslated region of the clone (FIG. 2) which are believed to beinvolved in the destabilization of specific mRNAs [10].

Since the ING1 gene was originally isolated by subtractive hybridizationbetween normal and transformed epithelial cDNAs, the levels of ING1 mRNAexpression in different normal, breast cancer, and brain cancer celllines were examined. Results from Northern blot analysis show that ING1is expressed at considerably lower levels (approximately 2-8 fold asestimated by scanning densitometry) in BT-20, ZR-75, MDA-MB-435 andT-47D breast cancer cells compared to MDA-MB-468 and SK-BR-3 breastcancer cells and to normal Hs68 fibroblasts. Results from reversetranscription polymerase chain reaction (RT-PCR) showed that ING1 is notexpressed or is expressed at very low levels in glioblastomas,astrocytomas and meningiomas.

Isolation of a DNA fragment that was capable of inducing tumors, fociand growth in soft agar when expressed in the antisense orientation,suggested that the cellular role of p33^(ING1) is to negatively regulategrowth. To test this idea, part of the ING1 cDNA was cloned into themammalian expression vector pBK in the sense orientation (pING1-S). Thisconstruct and the plasmid vector, both of which contain neomycinresistance genes and a cytomegalovirus (CMV) promoter, were transfectedinto human breast cancer (Hs578T) and normal fibroblast (Hs68) cells.Following growth of the cells in antibiotic for 3 weeks, a large numberof stable transformants were recovered from cells transfected withvector (1+3), whereas very few colonies were visible in plates of cellstransfected with the sense orientation of ING1 cDNA (2+4) (FIG. 4a).

To corroborate the results of these chronic assays, the effect ofmicroinjecting these constructs, together with non-specific antibodiesinto fibroblasts was examined. Arrows in panels 1 and 3 of FIG. 4bidentify cells injected with sense (S) and antisense (αS) constructs,respectively, which were visualized by staining for the presence ofcoinjected non-specific antibodies using indirect immunofluorescence.

Arrows in panels 2 and 4 of FIG. 4b show that cells injected withpING1-S failed to incorporate bromodeoxyuridine (BrdU) (panel 2) over a36 hour time course after injection. In contrast, those injected withpING1-αS entered S phase (panel 4) as estimated by staining withanti-BrdU antibodies.

FIG. 4c shows the combined results of 5 separate experiments, whichindicated that injection of the pBK vector or of pING1-αS constructs hadno appreciable effect upon the ability of injected cells to incorporateBrdU, whereas injection of pING1-S blocked the ability of cells to enterinto and proceed through S phase.

Similar results were obtained in larger populations of cells that wereelectroporated with vector, sense and antisense construct DNAs togetherwith a construct encoding the CD20 surface marker. Such co-transfectionsallowed the analysis of DNA content in cells that had taken up DNA bystaining for CD20 and subsequent analysis by fluorescence activated cellsorting (FACS). As shown in Table 2, the CD20-expressing populationco-transfected with pING1-S had, on average, 63.1% of cells in G0/G1whereas those co-transfected with vector had 33.6k of cells in G0/G1when cells were fixed and stained 48 hours after electroporation.

These results, using several independent methods, indicate that theoverexpression of p33^(ING1) inhibits cell growth and DNA synthesis inboth transient and chronic assays, most likely by arresting cells in theG1 phase of the cell cycle.

Since the activity of the tumor suppressor genes increases in senescentcells [20], p33^(ING1) activity in low and high passage cells waschecked. As shown in FIGS. 8a and 8b, ING1 expression (and the level ofthe p33^(ING1) protein) increased several-fold when cells approached theend of their in vitro replicative lifespan.

These data demonstrate that p33^(ING1) is a novel inhibitor of cellgrowth and a candidate tumor suppressor. Additional experiments alsoindicate that p33^(ING1) is localized in the nucleus of cells, which isconsistent with p33^(ING1) functioning as a tumor suppressor. Furtherdata showed that p33^(ING1) is localized to the 13q33-34 chromosomeregion. A number of human cancers have been mapped to this regionincluding primary gastric cancer; haematologic neoplasms; head and necksquamous cell carcinomas. Accordingly, it is contemplated that thenucleic acid sequences of the present invention may be used to detectcancerous or neoplastic cells of these types.

Alternatively, p33^(ING1) might play a role in the regulation ofcyclin-dependent kinases (CDKs), as reported recently for the family ofCDK inhibitors including p18[11], p21[12,13] and the candidate tumorsuppressor p16^(MTS1) [8] to which a portion of the p33^(ING1) sequenceshows some homology, and which has been reported to be the MTS1 multipletumor suppressor locus of human chromosome 9p21 that is inactivated inmany types of human tumors [14,15].

It is expected that several p33^(ING1) -related peptides will be usefulin the-present invention. In particular, p33^(ING1), its analogs andrelated proteins and peptides which are effective in suppressing theproliferation of cancerous cells are preferred.

Included within the scope of the p33^(ING1), as that term is usedherein, are p33^(ING1) s having the amino acid sequence set forth inFIGS. 2 and 3, glycosylated or deglycosylated derivatives of p33^(ING1),homologous amino acid sequence variants of the sequence of FIGS. 2 and3, and homologous in vitro-generated variants and derivatives ofp33^(ING1), which are capable of exhibiting a biological activity incommon with the p33^(ING1) of FIG. 3.

p33^(ING1) biological activity is defined as either: (1) immunologicalcross-reactivity with at least one epitope of native p33^(ING1), or (2)the possession of at least one cell proliferation, cell regulatory ortumor suppressive function qualitatively in common with nativep33^(ING1) s. One example of the qualitative biological activity ofp33^(ING1) is its ability to inhibit cell growth as estimated bydecreasing the S-phase fraction in a population of cells.

Immunologically cross-reactive, as used herein, means that the candidatepolypeptide is capable of competitively inhibiting the qualitativebiological activity of the native p33^(ING1) having this activity, withpolyclonal antisera raised against the known active analog. Suchantisera are prepared in conventional fashion by injecting goats orrabbits, for example, subcutaneously with the known active analog incomplete Freund's adjuvant, followed by booster intraperitoneal orsubcutaneous injection in incomplete Freunds.

This invention is concerned with amino acid sequence variants of nativep33^(ING1). Amino acid sequence variants of the p33^(ING1) are preparedwith various objectives in mind, including increasing the affinity ofthe p33^(ING1) for its binding partner, facilitating the stability,purification and preparation of the p33^(ING1), modifying its biologicalhalf-life, improving therapeutic efficacy, and lessening the severity oroccurrence of side effects during therapeutic use of the p33^(ING1).

Amino acid sequence variants of the p33^(ING1) fall into one or more ofthree classes: insertional, substitutional, or deletional variants.These variants ordinarily are prepared by site specific mutagenesis ofnucleotides in the DNA encoding the p33^(ING1), by which DNA encodingthe variant is obtained, and thereafter expressing the DNA inrecombinant cell culture. However, variant p33^(ING1) fragments havingup to about 100 to 150 amino acid residues are prepared conveniently byin vitro synthesis.

The amino acid sequence variants of the p33^(ING1) are predeterminedvariants not found in nature or naturally occurring alleles. Thep33^(ING1) variants typically exhibit the same qualitative biologicalactivity as naturally occurring p33^(ING1). However, the p33^(ING1)variants and derivatives that are not capable of exhibiting qualitativebiological activity similar to native p33^(ING1), may nonetheless beuseful as reagents in diagnostic assays for p33^(ING1) or antibodies top33^(ING1). Further, when insolubilized in accordance with knownmethods, they may be used as agents for purifying anti-p33^(ING1)antibodies from antisera or hybridoma culture supernatants. Further,they may be used as immunogens for raising antibodies to p33^(ING1) oras a component in an immunoassay kit (labeled so as to be a competitivereagent for native p33^(ING1) or unlabeled so as to be used as astandard for the p33^(ING1) assay) so long as at least one p33^(ING1)epitope remains active in these analogs.

While the site for introducing an amino acid variation may bepredetermined, the mutation, per se, need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random or saturation mutagenesis (where all 20 possible residuesare inserted) is conducted at the target codon and the expressedp33^(ING1) variant is screened for the optimal combination of desiredactivities. Such screening is within the ordinary skill of the art.

Amino acid insertions will usually be on the order of from about one toabout ten amino acid residues; substitutions are typically introducedfor single residues and deletions will range from about one to aboutthirty residues. Deletions or insertions preferably are made in adjacentpairs. That is, a deletion of two residues or insertion of two residues.Substitutions, deletions, insertions or any combination thereof may beintroduced or combined to arrive at a final construct.

Insertional amino acid sequence variants of the native p33^(ING1) arethose in which one or more amino acid residues extraneous to nativep33^(ING1) are introduced into a predetermined site in the targetp33^(ING1) and which displace the pre-existing residues. Commonly,insertional variants are fusions of heterologous proteins orpolypeptides to the amino or carboxyl terminus of the p33^(ING1). Suchvariants are referred to as fusions of the p33^(ING1) and a polypeptidecontaining a sequence which is other than that which is normally foundin the p33^(ING1) at the inserted position. Several groups of fusionsare contemplated for carrying out the invention described herein.

Immunologically active p33^(ING1) derivatives and fusions comprise thep33^(ING1) and a polypeptide containing a non-p33^(ING1) epitope. Suchimmunologically active derivatives and fusions of p33^(ING1) are withinthe scope of this invention. The non-p33^(ING1) epitope may be anyimmunologically competent polypeptide, i.e., any polypeptide which iscapable of eliciting an immune response in the animal in which thefusion is to be administered, or which is capable of being bound by anantibody raised against the non-p33^(ING1) polypeptide.

Substitutional variants are those in which at least one residue in theFIG. 3 sequence has been removed and a different residue inserted in itsplace. Novel amino acid sequences as well as isosteric analogs (aminoacid or otherwise) are included within the scope of this invention.

Some deletions, insertions and substitutions will not produce radicalchanges in the characteristics in the p33^(ING1) molecule. However,while it is difficult to predict the exact effect of the substitution,deletion or insertion in advance of doing so, for example, whenmodifying an immune epitope on the p33^(ING1) protein, one skilled inthe art will appreciate that the effect will be evaluated by routinescreening assays. For example, a change in the immunological characterof the p33^(ING1) protein, such as affinity for a given antibody, ismeasured by a competitive-type immunoassay. Modifications of proteinproperties such as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, or the tendency to aggregatewith carriers or into multimers may be assayed by methods well known toone of skill in the art.

Deletions of cysteine or other labile amino acid residues may also bedesirable. For example, they may increase the oxidative stability of thep33^(ING1) protein. Deletion or substitution of potential proteolysissites, e.g., Arg Arg, is accomplished by deleting one of the basicresidues or substituting one with glutaminyl or histidyl residues.

Covalent modifications of the p33^(ING1) protein are included within thescope of the present invention. Such modifications are introduced byreacting targeted amino acid residues with an organic derivatizing agentthat is capable of reacting with selected side chains or terminal aminoacid residues. The resulting covalent derivatives of p33^(ING1) areuseful to identify residues important for p33^(ING1), s biologicalactivity, for immunoassays of the p33^(ING1) or for preparation ofanti-p33^(ING1) antibodies for immunofinity purification of recombinantp33^(ING1). Such modification are within the ordinary skill of the artand are performed without undue experimentation.

In general, prokaryotes are used for cloning of DNA sequences and inconstructing the vectors useful in the present invention. For example,E. coli HB101, DH5α and XL1-blue are particularly useful. These examplesare meant to be illustrative and do not limit the present invention.Alternatively, in vitro methods of cloning such as the polymerase chainreaction may be used.

Expression hosts typically are transformed with DNA encoding thep33^(ING1) protein which has been ligated into an expression vector.Such vectors ordinarily carry a replication site, although this is notnecessary where chromosomal integration will occur. Expression vectorsmay also include marker sequences which are capable of providingphenotypic selection in transformed cells. Expression vectors alsooptimally will contain sequences which are useful for the control oftranscription and translation.

Expression vectors used in eukaryotic host cells will also containsequences necessary for the termination of transcription which mayaffect mRNA expression. Expression vectors may contain a selection geneas a selectable marker. Examples of suitable selectable markers formammalian cells are dihydrofolate reductase, thymidine kinase, neomycinor hygromycin.

Antibodies to the p33^(ING1) may be prepared in conventional fashion[18] by injecting goats or rabbits, for example, subcutaneously with thecomplete p33^(ING1) protein or a peptide consisting of at least 10 aminoacids similar to the p33^(ING1) protein in complete Freund's adjuvant,followed by booster intraperitoneal or subcutaneous injection inincomplete Freund's adjuvant, The anti-p33^(ING1) antibodies may bedirected against one or more epitopes on p33^(ING1). Monoclonalantibodies against p33^(ING1) can be prepared by methods known in theart [18]. The antibodies are preferably labelled with a marker, forexample, with a radioactive or fluorescent marker. It is contemplatedthat the antibodies would be labelled indirectly by binding them to ananti-goat or anti-rabbit antibody covalently bound to a marker compound.

C. Pharmaceutical Compositions

The present invention may be used to block the growth or decrease theproliferation of cancer cells by increasing expression of p33^(ING1).Blocking the growth of cancer cells is of obvious importance. A methodof inhibiting cell division, particularly cell division which wouldotherwise occur at an abnormally high rate, is also possible. Forexample,. increased cell division is reduced or prevented by introducinginto cells a drug or other agent which can increase, directly orindirectly, expression of p33^(ING1).

In one embodiment the p33^(ING1) protein or a peptide having p33^(ING1)biological activity is introduced directly. In a preferred embodimentthe peptide possesses at least one cell proliferation, cell regulatoryor tumor suppressive function qualitatively in common with nativep33^(ING1).

In another embodiment nucleotides coding for p33^(ING1) are introducedby retroviral or other means. In one embodiment the nucleotide codingfor p33^(ING1) comprises a nucleotide sequence which codes for the aminoacid sequence of p33^(ING1) as set forth in FIG. 3. In anotherembodiment the nucleotide sequence coding for p33^(ING1) comprises anucleotide sequence which codes for the amino acid sequence set forth inFIG. 2. Preferably the nucleotide sequence is substantially identical tothe cDNA sequence of FIG. 3, more preferably the sequence issubstantially identical to the cDNA sequence, of FIG. 2 and mostpreferably the sequence is substantially identical to nucleotides 161 to1143 of the cDNA sequence of FIG. 3.

Cell division is increased by preventing transcription of ING1 DNAand/or translation of RNA. This can be carried out by introducingantisense oligonucleotides of the ING1 sequence into cells, in whichthey hybridize to the p33^(ING1) -encoding mRNA sequences, preventingtheir further processing. It is contemplated that the antisenseoligonucleotide can be introduced into the cells by introducingantisense single-stranded nucleic acid which is substantially identicalto the complement of the cDNA sequence in FIGS. 2 or 3. It is alsocontemplated that an antisense oligonucleotide can be expressed in thecells by introducing a single- or double-stranded polynucleotide intothe cell under conditions wherein a single-stranded nucleic acidsequence which is substantially identical to the complement of the cDNAsequence in FIGS. 2 or 3 is expressed in the cell, for example, byplacing the polynucleotide in the antisense direction under the controlof a strong promoter. It is contemplated that the antisenseoligonucleotide introduced to the cell or expressed in the cell is atleast 100 nucleotides, more preferably it is at least 200 nucleotidesand most preferably it is at least 400 nucleotides in length. Mostpreferably the antisense oligonucleotide sequence is substantiallyidentical to the complement of nucleotides 942 to 1124 of the cDNAsequence set forth in FIG. 3.

It is also possible to inhibit expression of p33^(ING1) by the additionof agents which degrade p33^(ING1). Such agents include a protease orother substance which enhances p33^(ING1) breakdown in cells. In eithercase the effect is indirect, in that less p33^(ING1) is available thanwould otherwise be the case.

Viral or plasmid vectors may be used to deliver sense and antisenseconstructs to target cells in vivo. Such viral vectors may includeretroviruses, adenovirus or adenovirus-associated viruses. Such methodsare known in the art [19].

Parenteral administration of the nucleic acids is preferred withsubdermal or intramuscular administration most preferred. Intravenousadministration or use of implanted milliosmol pumps (available fromAlza) may also be used.

When used for parenteral administration, which is preferred, the nucleicacids of the present invention may be formulated in a variety of ways.Aqueous solutions of the nucleic acids of the present invention may beencapsulated in polymeric beads, liposomes, nanoparticles or otherinjectable depot formulations known to those of skill in the art.(Examples thereof may be found, for example, in Remington'sPharmaceutical Sciences, 18th Edition, 1990.) The nucleic acids may alsobe encapsulated in a viral coat. Doses are selected to provide effectiveinhibition of cancer cell growth and/or proliferation.

The methods of this invention may also be achieved by using apharmaceutical composition comprising one or more of the followingcancer cell proliferation inhibiting compounds: p33^(ING1), its analogsand related proteins and peptides. Doses are selected to provideeffective inhibition of cancer cell growth and/or proliferation.

Parenteral administration of the proteins or peptides is preferred, withsubdermal or intramuscular administration most preferred. Intravenousadministration or use of implanted milliosmol pumps (available fromAlza) may also be used.

When used for parenteral administration, which is preferred, theproteins and peptides of the present invention may be formulated in avariety of ways. Aqueous solutions of the proteins or peptides of thepresent invention may be encapsulated in polymeric beads, liposomes,nanoparticles or other injectable depot formulations known to those ofskill in the art. (Examples thereof may be found, for example, inRemington's Pharmaceutical Sciences, 18th Edition, 1990.)

Compositions including a liquid pharmaceutically inert carrier such aswater may also be considered for both parenteral and oraladministration. Other pharmaceutically compatible liquids may also beused. The use of such liquids is well known to those of skill in theart. (Examples thereof may be found, for example, in Remington'sPharmaceutical Sciences, 18th Edition, 1990.)

The dose level and schedule of administration may vary depending on theparticular p33^(ING1) -related compound(s) and/or compositions used, themethod of administration, and such factors as the age and condition ofthe subject.

As discussed previously, parenteral administration is preferred, butformulations may also be considered for other means of administrationsuch as orally, per rectum, and transdermally. The usefulness of theseformulations may depend on the particular compound used and theparticular subject receiving the p33^(ING1) -related compound.

Oral formulations of p33^(ING1) -related compounds may optionally andconveniently be used in compositions containing a pharmaceutically inertcarrier, including conventional solid carriers, which are convenientlypresented in tablet or capsule form. Formulations for rectal ortransdermal use may contain a liquid carrier that may be oily, aqueous,emulsified or contain certain solvents suitable to the mode ofadministration. Suitable formulations are known to those of skill in theart. (Examples thereof may be found, for example, in Remington'sPharmaceutical Sciences, 18th Edition, 1990.)

D. Use of ING1 DNA and RNA and p33^(ING1) and Related Proteins andPeptides for Diagnosis

The present invention also has diagnostic use, since simpleimmunochemical staining of cells or sections of cells should give anaccurate estimate of the portion of cells expressing p33^(ING1). Such atest based on the use of anti-p33^(ING1) antibodies or ING1polynucleotides and other standard secondary techniques of visualizationwill be useful in cancer diagnosis. Such a test of tumor suppressor geneexpression might also be useful to the scientific research community.

Antibodies specifically reactive with p33^(ING1) can be produced, usingknown methods [18]. For example, anti-p33^(ING1) antisera can beproduced by injecting an appropriate host (e.g., rabbits, mice, rats,pigs) with p33^(ING1) and withdrawing blood from the host animal aftersufficient time for antibodies to have been formed. Monoclonalantibodies can also be produced using known techniques [18]. Suchantibodies to p33^(ING1) will generally be detectably labelled (e.g.,with a radioactive label, a fluorescent material, biotin or anothermember of a binding.pair or an enzyme) by methods known in the art. Itis also contemplated that the anti-p33^(ING1) antibodies may beindirectly labelled by binding to another second antibody which secondantibody is detectably labelled.

In a diagnostic method of the present invention, cells obtained from anindividual or a culture are processed in order to determine the extentto which p33^(ING1) is present in cells, in a specific cell type or in abody fluid. This can be determined using known techniques and anantibody specific for p33^(ING1). Comparison of results obtained fromcells or a body fluid being analyzed with results obtained from anappropriate control (e.g., cells of the same type known to have normalp33^(ING1) levels or the same body fluid obtained from an individualknown to have normal p33^(ING1) levels) is carried out. Decreasedp33^(ING1) levels are indicative of an increased probability of abnormalcell proliferation or oncogenesis or of the actual occurrence ofabnormal proliferation or oncogenesis. It is contemplated that thelevels of p33^(ING1) in cancerous cells will be at least 50% less thanthe level of p33^(ING1) in non-cancerous cells, morepreferably thelevels will be less than 30% of normal levels, most preferablyp33^(ING1) will not be expressed.

It is contemplated that a diagnostic kit could include a solid supportfor attaching the cell or tissue to be tested and a detectably labelledanti-p33^(ING1) antibody. It is further contemplated that theanti-p33^(ING1) antibody may not be labelled but the kit wouldadditionally contain another detectably labelled antibody capable ofbinding to the anti-p33^(ING1) antibody.

A hybridization probe comprising RNA, ING1 cDNA or ING1 genomic DNAhaving a sequence substantially identical to FIG. 3 ("ING1polynucleotide") may be employed as a means for determining the sequenceof the ING1 gene present in the genomic DNA of a given sample, or thelevel of ING1 mRNA expressed in cells of such sample. Such hybridizationprobes will generally be detectably labelled (eg. with a radioactivelabel, a fluorescent label, biotin, etc). It is also contemplated thatthe ING1 polynucleotide may be indirectly labelled by methods known inthe art.

A tissue sample or cell sample can be prepared by conventional means andprobed with the labelled ING1 polynucleotide probe to determine thelevel of expression of ING1 mRNA in the cells. The ING1 polynucleotideprobe may also be used to determine whether the genomic DNA from thecell sample has a mutation or rearrangement of its ING1 gene by methodsknown in the art (i.e. PCR sequencing or restriction fragment lengthpolymorphism analysis). The polynucleotide probe may also be used todetermine whether the genomic DNA from the cell sample has amutation/deletion rearrangement of the chromosome region of 13q33-34.

The oligonucleotide probe useful in these methods may comprise at leastabout 20 nucleotides which sequence is substantially identical to thesequence of FIG. 3, more preferably it will comprise at least about 100nucleotides, and most preferably it will comprise at least 400nucleotides. In the case of PCR sequencing it is contemplated that oneof the two ING1 oligonucleotide primers will be substantially identicalto one region of the sequence of FIG. 3 and that the secondoligonucleotide primer will be substantially identical to the complementof a second region of the sequence of FIG. 3. The size of these primersis preferably from 5-25 nucleotides, more preferably from 10-20nucleotides. Most preferably the oligonucleotide probes and primers willbe substantially identical to the coding region of the cDNA sequence ofFIG. 3. Such nucleotides can be generated synthetically by conventionalmeans.

Comparison of the results obtained from cells or a body fluid beinganalyzed with results obtained from an appropriate control (eg. cells ofthe same type known to have abnormal or native p33^(ING1) or fluid froman individual known to have normal p33^(ING1)) is carried out. DecreasedING1 mRNA levels are indicative of an increased probability of abnormalcell proliferation or oncogenesis or of the actual occurrence ofabnormal proliferation or oncogenesis. It is contemplated that thelevels of ING1 mRNA in cancerous cells will be at least 50% less thanthe level of ING1 mRNA in non-cancerous cells, more preferably thelevels will be less than 30% of normal levels, most preferably ING1 mRNAwill not be expressed.

The presence of a mutation/deletion in one copy of the ING1 gene in adiploid cell is also indicative of an increased probability thatabnormal cell proliferation or oncogenesis will occur. The presence ofmutations/deletions in both copies of the ING1 gene is indicative ofpossible or actual oncogenesis.

The following examples are offered to illustrate this invention and arenot meant to be construed in any way as limiting the scope of thisinvention.

EXAMPLES

The methods described as follows were used to perform the studiesdescribed herein. In addition, the generally known methods set forth inlaboratory manuals for molecular cloning and antibody techniques [e.g.,17,18] may advantageously be used by one of skill in the art to produceadditional embodiments of the invention.

Example 1 Strategy for Cloning and Biological Assays

Subtractive hybridization of breast cancer cell line cDNAs with CDNAfrom normal mammary epithelial cells, subcloning of subtracted cDNAsinto the PLNCX retroviral vector [7] and injection into nude mice wasdone essentially as described [3] with the modifications noted below.The cloning of full length cDNA was done using standard methods [17].The strategy is shown in FIG. 1a.

cDNA was prepared from an non-transformed mammary epithelial cell line(184A1) [6] and digested with the restriction enzyme Sau3A. cDNAs fromthe breast cancer cell lines MCF-7, BT-483, BT-474, Hs-578T, ZR-75,MD-MB-468, MD-MB-435 and BT-20 (obtained from the American Type CultureCollection, Bethesda Md.) were also digested with Sau3A. Fragments oftester DNA (cDNA from normal epithelial cells) were ligated to "a"adaptors. Fragments of driver DNA (cDNA from breast tumor cells) wereligated to "b" adaptors. Adaptors were prepared by annealing thesynthetic oligonucleotides:

5'-GACCTGGCTCTAGAATTCACGACA-3' (SEQ ID NO: 3) with

5'-GATCTGTCGTGAATTCTAGAGCCAGG-3' (SEQ ID NO: 4) (adaptor "a"); and

5'-GACTCGACGTTGTAACACGGCAGT-3' (SEQ ID NO: 5) with

5'- GATCACTGCCGTGTTACAACGTCGAG-3' (SEQ ID NO: 6) (adaptor "b").

The mixture of driver DNA and tester DNA was denatured, then hybridizedat 66° C. for 18 hours. After hybridization, mixtures were treated withMung bean nuclease to eliminate single-stranded adaptor-derived endsfrom "heterozygous" hybrids (hybrids containing both a and b adaptors).Resultant double-stranded molecules were then selectively amplified byPCR using primer "a".

The "amplicons" were then subjected to five successive rounds ofhybridization, selective degradation and PCR amplification using 40 μgof driver cDNA containing adaptors and 200 ng, 5 ng and 5 pg of testeramplicons in respective rounds. This procedure allowed for a significantenrichment of sequences that were more highly expressed in thephenotypically normal epithelial cells as determined by slot blothybridization.

All subtracted fractions were combined and used as a probe to screen anear-senescent human diploid fibroblast cDNA library. Followingscreening, 300 cDNA clones were isolated and their inserts were randomlyfragmented (200-800 bps). These were then ligated with adaptors preparedby annealing two oligonucleotides 5'-AATCATCGATGGATGGATGG-3' (sense)(SEQ ID NO: 22) 5'-CCATCCATCCATCGATGATTAAA-3' (SEQ ID NO: 23) and wereamplified by PCR using the "sense" strand of the adaptor as the PCRprimer. PCR amplified DNA was recloned into the ClaI site of theretroviral plasmid vector pLNCX [7] with synthetic adaptors carryinginitiation codons in all reading frames. This library of about 10⁵clones, enriched in tumor suppressor sequences was then used for theisolation of transforming genetic suppressor elements (GSEs).

After transfection of the recombinant retroviral plasmids into thepackaging line BOSC 23 [25], retroviruses containing the isolated cDNAfragments were used to infect non-tumorigenic immortalized mouse mammaryepithelial cells (NMuMG) which were subsequently injected subcutaneouslyinto nude mice. Subcloning into the retroviral vector, packaging intothe BOSC 23 virus-packaging cell line and assays using nude mice wereperformed as described [3].

After 45 days, two mice developed tumors from which two cDNA insertswere recovered by PCR, one of which is subsequently shown to beexpressed in the antisense orientation. The primers used in the PCRamplification were:

5'-CCAAGCTTTGTTTACATCGATGGATG-3' (SEQ ID NO: 7) (sense); and

5'-ATGGCGTTAACTTAAGCTAGCTTGCCAAACCTAC-3' (SEQ ID NO: 8) (antisense). Therecovered cDNA insert which was in the antisense orientation wasdigested with ClaI and HindIII and recloned back into the retroviralvector, pLNCX, in the same position and orientation and then testedindividually in vitro.

NMuMG cells were infected with retrovirus produced from pLNCX vectorcontaining or not containing the ING1 insert (nucleotides 942 to 1,124of the ING cDNA set forth in FIG. 3). The soft agar culture wascomprised of two layers: an underlay (DMEM, 10% FCS, 0.6% agar) and anoverlay (DMEM, 10% FCS, 0.3% agar). 5×10⁴ cells were plated in soft agarin 10 cm plates and were left at 37° C. for 6-7 weeks before beingcounted. 5×10⁵ transfected NIH 3T3 cells were plated per 10 cm dish.Transfected NIH 3T3 cells were grown in 5% serum for 4 weeks prior tofixing and visualizing foci. pLNCX-S and pLNCX-αS represent sense andantisense orientations of the ING1 cDNA insert, respectively.

                                      TABLE 1                                     __________________________________________________________________________    Results of the soft agar and focus forming assays                                     Soft agar assay     Focus forming assay                               Trail Number                                                                          1   2   3   4   mean                                                                              1  2  mean                                        __________________________________________________________________________    pLNCX (vector)                                                                        0   0   0   0   0   9  13 11                                          pLNCX-Ras                                                                             224 248 208 (--)                                                                              226.7                                                                             (--)                                                                             (--)                                                                             (--)                                        pLNCX-αS                                                                        42  46  41  82  52.8                                                                              18 34 26                                          pLNCX-S (--)                                                                              (--)                                                                              0   0   0   (--)                                                                             (--)                                                                             (--)                                        __________________________________________________________________________     (--) = not determined                                                    

These results showed that the antisense ING1 cDNA insert causedincreased cell proliferation.

Panel 1 of FIG. 1b shows NMuMG cells infected with the retroviral vectorpLNCX and panel 2 of FIG. 1b shows cells infected with the retroviralvector pLNCX containing the antisense ING1 insert. The bar equals 1 mm.Panel 3 of FIG. 1b shows NIH 3T3 cells transfected with vector alone andpanel 4 of FIG. 1b shows cells transfected in parallel with pLNCXcontaining the antisense ING1 insert.

Example 2 cDNA of ING1 and Predicted Amino Acid Secuence of p33^(ING1)

In order to isolate the gene corresponding to the fragment showingbiological effects, normal human fibroblast and HeLa cell cDNA librarieswere screened with the ING1 cDNA fragment from Example 1, resulting inthe isolation of 11 positive clones. Two clones containing the largestcDNA inserts were sequenced on both strands using an Applied Biosystemsautomated sequencer, yielding the sequence shown in FIG. 2.

In order to obtain the 5' end of the ING1 gene, 5' RACE (rapidamplification of cDNA ends) was used. Total cDNAs isolated followingreverse transcription had the synthetic adaptor5'-GTACATATTGTCGTTAGAACGCGTAATACGCCTCACTATAGGGA-3' (SEQ ID NO: 11)ligated to them and PCR reactions using nested primers from both theadaptor and the ING1 gene were used to amplify the 5' ends of allRT-generated cDNAs. The primers used in the amplification were:5'-CTGGATCTTCTCGTCGCC-3' (SEQ ID NO: 12) and 5'-AGTGCAGCATCGGCCGCTTC-3'(SEQ ID NO: 13) from the ING1 sequence and:5'-GTACATATTGTCGTTAGAACGCG-3' (SEQ ID NO: 14) and5'-TAATACGCCTCACTATAGGGA-3' (SEQ ID NO: 15) from the adaptor sequence.The largest PCR products were recovered from agarose gels followingelectrophoresis and were subcloned and sequenced to generate thefull-length sequence shown in FIG. 3.

The predicted coding region of ING1 begins at nucleotide 16 and ends atnucleotide 898, as shown in FIG. 3, predicting a translation product of33,350 daltons. Comparison of the sequence of p33^(ING1) to theavailable protein and nucleotide data bases showed no significanthomology to any sequence encoding a known protein and very limitedsimilarity to retinoblastoma binding protein 2 (RbBP2) [9] and toseveral zinc finger transcription factors. Regions of the p33^(ING1)protein that show homology to different members of the p16^(MTS1) familyof cyclin-dependent kinase inhibitors and to retinoblastoma bindingprotein 2 were identified using the Blast program available from theNational Centre for Biological Information (address:www.ncbi.nim.nih.gov).

Example 3 Expression of a GST-p33^(ING1) Fusion Protein and Creation ofanti-p33 Polyclonal Antibody

In order to generate polyclonal antibodies, a fragment of ING1containing nucleotides 161-1146 of FIG. 3 was subcloned into theEcoRI-Xhol sites of the bacterial expression vector pGEX-4T1 (PharmaciaBiotech, Inc., Quebec, Canada) containing the glutathione-bindingportion of glutathione-S-transferase (GST). Plasmids were sequenced toverify that the correct reading frame was obtained and the constructswere electroplated into E.coli XL1-Blue. Following selection, bacterialcultures were induced to express the fusion protein by the addition of0.1 mM isopropyl thio-galactopyranoside (IPTG) and fusion protein waspurified by standard glutathione-Agarose column affinity chromatography.Eluted GST-p33^(ING1) fusion protein was dialyzed and used in immunogenin female New Zealand white rabbits. After four boosters, rabbits werebled and their serum tested for reactivity against the fusion protein.All animals showed reactivity and the bleeds showing the highest titerwere chosen for subsequent use in Western blot, immunoprecipitation andimmunofluorescence protocols.

Example 4 Effect of the Antisense ING1 Fragment on Expression ofp33^(ING1) in Tissue Culture Cells

Analysis of p33^(ING1) protein levels in cell samples was performed byWestern blotting using anti-p33^(ING1) antibodies raised against theGST-p33^(ING1) fusion protein. Proteins were separated byelectrophoresis in 12.5% polyacrylamide/SDS gels, andelectrophoretically transferred to membranes for 1 hour. The membraneswere blocked in TBS (100 mM Tris, 150 mM NaCl) containing 10% non-fatdried milk and 0.1% Tween-20, for 2 hours. Incubation of the membraneswith p33^(ING1) antiserum was performed in TBS containing 5% nonfat milkand 0.1% Tween 20 (TBST) for 30 minutes. Horseradishperoxidase-conjugated goat anti-rabbit antibody was then applied to thefilters for 1 hour in TBST. Peroxidase activity was detected using achemiluminescence system (Amersham Canada, Oakville Ontario Canada)

As shown in FIG. 1c, NMuMG (lane 1) and ZR-75 (lane 2) cell lines weretested. The Western blot analysis of human and mouse cell lysatesrevealed a protein of 33 kD. Preincubation of antibodies withGST-p33^(ING1) fusion protein blocked recognition of p33^(ING1) in aparallel blot using lysates from the same cells (lanes 3 and 4).

To determine whether the level of p33^(ING1) was decreased in cellsinfected with viral constructs containing the antisense orientation,lysates were prepared from control NMuMG cells and from NMuMG cellsinfected with antisense ING1 (pLCNX-αS) that had grown and formedcolonies in semi-solid medium. A Western blot of lysates from NMuMGcells infected with pLNCX vector (lane 5) or pLNCX vector containingantisense ING1 insert (lane 6) by the method set out above is shown inFIG. 1C. Results of the Western blot analysis showed that chronicexpression of antisense construct reduced the expression of theendogenous ING1 gene by approximately 90% compared to control parentalcells.

Example 5 Effects of p33^(ING1) Overexpression

Isolation of a DNA fragment that was capable of inducing foci and growthin soft agar when expressed in the antisense orientation, suggested thatthe cellular role of ING1 might be to negatively regulate growth. Totest this idea, part of the ING1 cDNA (basepairs 161 to 1143 of FIG. 3)was cloned into the mammalian expression vector pBK (Stratagene, Aurora,Ontario Canada) in the sense orientation (pING1-S). This construct andthe plasmid vector, both of which contain neomycin resistance genes anda cytomegalovirus (CMV) promoter, were transfected into human breastcancer (Hs578T) and normal fibroblast (Hs68) cells. Following growth for3 weeks in medium containing G418, plates were fixed and stained withCoomassie Brilliant Blue to identify surviving colonies. A large numberof stable transformants were recovered from cells transfected withvector whereas very few colonies were visible in plates of cellstransfected with the sense orientation of the cDNA of ING1. FIG. 4Ashows the results when human Hs578T breast cancer cells (panels 1 and 2)and normal fibroblasts (panels 3 and 4) were transfected with theplasmids pBK (panels 1 and 3) or pING1-S containing the ING1 cDNA in thesense orientation (panels 2 and 4).

In order to corroborate the results of these chronic assays, we nextexamined the effect of microinjecting these constructs on the ability ofnormal diploid fibroblasts to initiate DNA synthesis.

Hs68 cells were plated on glass coverslips, deprived of serum for 12hours, microinjected with the indicated mixture of plasmid DNA (0.1μg/ml) plus nonspecific IgG (2 μg/ml) and were then incubated for 36hours in complete medium containing BrdU. Fixed cells were identified bystaining for injected IgG and for the incorporation of BrdU.Microinjection, fixation and staining were done as described previously[16].

FIG. 4C shows the combined results of 5 separate experiments. Each grouprepresents 110-200 injected cells. As shown in FIG. 4B, normal Hs68 HDFswere injected with solutions containing pING1-S plus non-specific rabbitIgG (panels 1 and 2) or with pING1-αS containing the ING1 cDNA in theantisense orientation plus non-specific rabbit IgG (panels 3 and 4).Injected cells were grown in the presence of BrdU and were fixed andstained for the presence of co-injected IgG (panels 1 and 3) orincorporated BrdU (panels 2 and 4). Arrows identify injected cells.Arrows in panels 2 and 4 show that cells injected with pING1-S failed toincorporate bromodeoxyuridine (BrdU, panel 2) over a 36 hour time courseafter injection, whereas those injected with pING1-αS entered S phase(panel 4) as estimated by staining with anti-BrdU antibodies. FIG. 4Cshows the results of 5 separate experiments which indicate thatinjection of the pBK vector or of pING1-αS constructs had no appreciableeffect upon the ability of cells to proceed through S phase.

Similar results were obtained in larger populations of cells that wereelectroporated with vector, sense and antisense construct DNAs togetherwith a construct encoding the CD20 surface marker. Such co-transfectionsallowed the analysis by flow cytometry of DNA content in transfectedcells that were positive for CD20 staining. Hs68 cells wereco-transfected with pCMV-CD20 together with pBK-p33^(ING1) -S or withpBK vector as a negative control. Cells were fixed and stained for CD20expression using commercially available antibodies and with propidiumiodide 48 hours after electroporation. Cell cycle distribution wasdetermined by flow cytometry using fluorescence-activated cell sorting.The percentage of the CD20+ cells in different phases of the cell cycleis shown for two independent experiments.

                  TABLE 2                                                         ______________________________________                                        Overexpression of p33.sup.ING1 arrested cells in G0/G1                        pBK (vector)        pBK-ING1-S                                                G1/G0       S       G2/M    G1/G0  S     G2/M                                 ______________________________________                                        Trial 1                                                                              32.7     38.5    28.8  53.3   19.9  26.8                               Trial 2                                                                              34.5     35.9    29.6  72.8   19.7  7.5                                mean   33.6     37.2    29.2  63.1   19.8  17.2                               ______________________________________                                    

As shown in Table 2, the CD20-expressing population that wasco-transfected with pING1-S had, on average, 63.1% of cells in G0/G1whereas those co-transfected with vector had 33.6% of cells in G0/G1when cells were fixed and stained 48 hours after electroporation. Theseresults, using several independent methods, indicate that theoverexpression of ING1 inhibits cell growth in both transient andchronic assays, most likely be arresting cells in the G1 phase of thecell cycle.

Example 6 Alterations of ING1 in Cancer Cell Lines

Since ING1 was originally isolated by subtractive hybridization betweennormal and transformed epithelial cDNAs, the ING1 gene and itsexpression in breast cancer cell lines was also examined. In FIG. 5A,lane 1 is MCFICA phenotypically normal epithelial cell line from mammarygland; lane 2 is MDA-MB-468; lane 3 is ZR-75; lane 4 is BT-20; lane 5 isSK-BR-3; lane 6 is MCFT; lane 7 is Hs578T and lane 8 is BT-474 (breastcancer cell lines). FIG. 5B shows the coomassie-blue stained gelcorresponding to FIG. 5A. The expression of p33^(ING1) in the cell lineswas tested by preparing lysates of cell lines and Western blotting usinganti-p33^(ING1) antibodies by the method in Example 4. Although analysisof genomic fragments containing the ING1 gene did not reveal anystructural changes in breast cell lines, results from Western blotanalyses shown in FIG. 5 suggest that the p33^(ING1) protein wasexpressed at considerably lower levels in some breast cancer cellscompared to a phenotypically normal epithelial cell line. Thisobservation of reduced expression in the absence of mutation is similarto the expression of BRCA-1 reported to occur in non-hereditary forms ofbreast cancer [25].

Normal diploid control cell strains and neuroblastoma cell lines wereanalyzed by Western Blot analysis in a manner similar to that set forthfor the breast cancer cell lines. FIG. 6a illustrates the Westernblotting results of IMR-5 (lane 1) SK-L-C6 (lane 2); SK-N-SH (lane 3)all neuroblastoma cell lines, and W138 (lane 4) a normal diploid lungfibroblast cell line. Normal diploid fibroblast cells expressed lowlevels of p33^(ING1) while immortalized neuroblastoma cells expressedconsiderably higher levels and in the case of the SK-N-SH neuroblastomaline a truncated protein was observed.

To investigate the nature of the change(s) responsible for truncatingp33^(ING1) in this neuroblastoma cell line, two complementary approacheswere taken. Southern blot analysis of DNA, from neuroblastomas and fromnormal fibroblasts that was digested with different restrictionendonucleases and probed with a ING1 nucleic acid probe, clearlyindicated that p33^(ING1) was rearranged in the neuroblastoma cell line.Human genomic DNAs were digested with HindIII, DraI or Pst1,electrophoresed through a 0.7% agarose gel, transferred to anitrocellulose membrane and hybridized with [³² P]-labelled p33^(ING1)cDNA. Hybridization was performed using standard procedures [17]. Lanes1-6 show the results for neuroblastoma SK-N-SH (2, 4 and 6) and fornormal diploid W138 cells (1, 3 and 5). Patterns such as those shown byW138 cells were also seen in other normal diploid cell strains.

To confirm that changes in the p33^(ING1) gene had occurred in theneuroblastoma cell line and to determine their nature by an independentmethod, reverse transcription polymerase chain reaction (RT-PCR) withRNA from SK-N-SH neuroblastoma cell line and from a phenotypicallynormal epithelial cell line (MCF-10) was performed as described [20].Neuroblastoma cDNA was amplified with PCR primers specific for the p33gene (direct (d) and reverse (r) primers). These are numbered and shownunderlined in FIG. 3 and the PCR products were compared with PCRfragments generated in parallel from control cell cDNA. FIG. 6c, lanes 1(primers 1d-4r) , 3 (1d-2r), 5 (2d-4r) and 7 (2d-3r) show the resultsfor W138, and lanes 2 (1d-4r), 4 (1d-2r), 6 (2d-4r) and 8 (2d-3r) showthe results for the neuroblastoma cell line. Primers were 1d:GTAGCGCAGTCTGACAAGCC (nucleotides 474-494 of SEQ ID NO: 9) 2d:TGGTTCCACTTCTCGTGCGT (763-782 of SEQ ID NO: 9) 2r: ACGCACGAGAAGTGGAACCA(SEQ ID NO: 16) 3r: TTTGGATTTCTCCAGGGCTT (SEQ ID NO: 17) and 4r:TACCTGTTGTAAGCCCTCTC (SEQ ID NO: 18). M shows a 1 kb ladder moleculeweight marker. All primer pairs gave similar results in both cell linesexcept for those using primers beyond nucleotide 858. For example, usingprimers 3r and 4r give no PCR product using neuroblastoma cDNA which isconsistent with data indicating that a deletion or a rearrangement hadoccurred within the p33^(ING1) gene. These experiments corroborate theidea that the 3' region of the p33^(ING1) gene was mutated in thisneuroblastoma.

Normal diploid control cell strains and brain cancer cell lines wereanalyzed by RT-PCR analysis. Reverse transcription with total RNA fromeach of the cell lines was performed by the method set out in Example 9.The same primer pairs set forth in Example 9 were used. FIG. 7illustrates the RT-PCR results of glioblastoma (lanes GB1-GB4)astrocytoma (lanes AS1-AS3) and meningioma (MN1-MN3) as compared to acontrol cell line (C114 C2). The ING1 mRNA was expressed at considerablylower levels, or not expressed at all, in the glioblastomas,astrocytomas and meningiomas as compared to the normal cell line.

Example 7 Nuclear Localization of p33^(ING1)

The experiments described below were performed with a rabbit polyclonalantibody (αp33) which was raised against a bacterially expressedglutathione-S-transferase (GST)-p33^(ING1) fusion protein and whichreacted with a 33 kDa protein in human and mouse cell Iysates asprepared by the method in Example 3.

In the first series of experiments, we determined the location ofp33^(ING1) in fibroblasts by examining the staining pattern of anti-p33antibody in fibroblasts by indirect immunofluorescence. For indirectimmunofluorescence normal human diploid fibroblasts (Hs68 cells) weregrown on glass coverslips for 48 hours at 37° C. to 60% confluence. Thecells were fixed in 3.7% formaldehyde, washed in 0.5% Triton X-100 andin 0.05% Tween 20 for 10 minutes each at room temperature. Formaldehydeand detergents were diluted in phosphate buffered saline (PBS) pH 7.5.The cells were incubated with a 1:100 dilution of rabbit p33^(1NGI)antiserum for 30 min, washed in PBS with 0.05% Tween, incubated withgoat anti-rabbit IgG-biotin antibody and then with streptavidinconjugated Texas Red [16]. Samples were examined with a Zeiss Axiophotfluorescence microscope and images were photographed on Kodak TMAX 100film.

Staining with polyclonal rabbit antibody alone was observed both innuclear and cytoplasmic compartments. Similar results were obtained withanti-p33 antibodies which were preincubated with 5 μg of GST proteinindicating that the signal was specific for p33^(ING1). When theanti-p33 serum was preincubated with 5 μg of GSTp33 fusion protein,nuclear staining was lost completely but cytoplasmic staining remained,indicating that the vast majority of p33^(ING1) staining was nuclear.

To confirm the nuclear localization of the 33 kDa protein. The pING1-sconstruct of Example 5 was microinjected into normal Hs68 fibroblastcells which were fixed and stained with anti-p33-antibody 24 hours afterinjection. Strong staining was clearly localized to the nucleus. Theseresults corroborate staining patterns in uninjected cells and show thatp33^(ING1) is localized primarily, and possibly exclusively, throughoutthe nucleoplasm.

Example 8 Chromosomal Localization of the ING1 gene

To identify the chromosomal localization of the ING1 gene, a genomic18-kb DNA insert containing the gene was labelled with digoxygenin-dUTPand hybridized to synchronized human lymphocyte metaphase spreads.

A genomic clone of the ING1 gene was isolated from a lambda FIX IIplacental human genomic library (Stratagene, Aurora, Ontario, Canada)with nucleotides 161 to 1143 of the ING1 sequence of FIG. 3 using highstringency (65° C. 0.133 SSC, 0.1% SDS) screening. The identity of theclone was confirmed by partial sequence analysis.

FISH was performed using established methods on methotrexate/thymidinesynchronized, phytohemagglutinin stimulated, normal peripheral bloodlymphocytes [2I]. Approximately 50 metaphase spreads were examined forprobe localization. Suppression for 30 minutes with a mixture ofsonicated human DNA (Sigma Diagnostics, Mississauga, Ontario, Canada)and cotl DNA (Gibco/BRL, Burlington, Ontario, Canada) was required toreduce the background. The stained slides were counterstained with DAPIand actinomycin D (for a DA-DAPI banding pattern) and were mounted inantifade medium and visualized utilizing a Zeiss Axioplan 2 microscope.Images of representative mitoses were captured using a cooled CCD camera(Photometrics PXL 1400). Digital alignment of the images from each flourwas done after registration calibration through a triple bandpass filter(FlTC/Texas Red/DAPI) to minimize registration error, utilizingcommercial software (Electronic Photography vl.3, Biological DetectionInc., Pittsburgh Pa.).

The results clearly showed localization of the probe to chromosomalregion 13q33-34. At least one specific probe signal was present in morethan 90% of the mitoses examined. Approximately 80% of the cells had twochromatids of a single chromosome. Approximately, 40% showed labellingof both chromatids of both chromosomes. More than 90% of the signalswere localized to a single band. In addition, cohybridization ofp33^(ING1) with a commercial 13/21 alpha-satellite probe (Oncor,Gaithersberg Md.) showed hybridization to the same chromosome.

The ING1 gene has been localized to an area near known sites of genomicalteration in several human cancers: primary gastric cancer [22],haematologic neoplasms [23] and head and neck squamous cell carcinomas[24].

Example 9 Expression Levels of ING1 in Young and Senescent Fibroblasts

The normal human diploid fibroblast cell strain Hs68 (ATCC CRL#1635) anda phenotypically normal mouse epithelial cell line from mammary gland(NMUNG) were grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum. Hs68 cells were used at 30 ("young"), 70 ("pre-aged") and 80 ("old") mean population doublings (MPDS) forexpression and life span experiments. After retroviral infection, thehuman diploid fibroblast cells (HDFs) were repeatedly passaged in 10 cmplates, splitting at a ratio of 1:2 when confluent.

For infection of fibroblasts, the retroviral vector (PLNCX) was used.The highly efficient ecotropic (BOSC23) and amphotropic (CAK8) packagingcell lines were used [26]. pLNCX-αS or pLNCX alone, were transfectedinto the BOSC23 virus-packaging cell line. Ecotropic and amphotropicpackaging lines, and the retroviral vector were kindly provided by Dr.A. Gudkov (University of Illinois at Chicago). The amphotropic cellswere infected by viruses from the BOSC23 supernatant. Fibroblasts wereplated at 10⁵ cells per 10 cm plate and infected with undiluted viralsupernatant from amphotropic producer cells. Infection efficienciesranged from 85 to 95% in individual trials.

Since the activity or expression levels of several tumor suppressorsincrease in senescent cells, the levels of ING1 expression in low andhigh passage cells were checked. All experiments were performed on theHs68 strain of primary normal human diploid fibroblasts. Senescent cellswere obtained by passaging early-passage ("young") fibroblastscontinuously to a point at which one population doubling required from2-3 weeks to complete compared to 24 hours, on average, for young HDFS.Hs68s at 80 MPDs exhibited characteristics typical of senescent cells,including an inability to respond to growth factors and alteredmorphology including increased size and decreased saturation density.

To study the level of expression of ING1 mRNA, RT-PCR using total RNAisolated from young and old cells was performed (FIG. 8A). The relativelevels of ING1 transcript were compared to the internal control geneglyceraldehyde-3-phosphate dehydrogenase (GAPDH) using PCR primersspecific for the p33 and GAPDH genes. ING1 and GAPDH were amplified inthe same reaction tube using the "primer dropping" approach [27] whichinternally controls for efficiency of reverse transcription andamplification by PCR.

Reverse transcription (RT) with 1 μg of total RNA from young and oldHs68 cells was performed using 50 U of RNasin (Pharmacia Biotech, Inc.,Quebec Canada) and 200 U of MMLV reverse transcriptase for 50 min. at42° C. in 20 μl reaction volumes. Two μl of each RT reaction wasamplified using 2 U of Taq polymerase. The two sets of primer pairs forthe ING1 gene and for the GAPDH gene that were used, were:5'-GAAGCGGCGGATGCTGCACT-3' (SEQ ID NO: 19); and5'-ACGCACGAGAAGTGGAACCA-3' (SEQ ID NO: 16) for the ING1 gene and 5'CGGAGTCAACGGATTTGGTCGTAT -3' (SEQ ID NO: 20); and 5'-AGCCTTCTCCATGGTGGTGAAGAC 3' (SEQ ID NO: 21) for the GAPDH gene. Thirtytwo PCR cycles for ING1 and twenty two PCR cycles for GAPDH wereperformed using standard conditions [17]. Primers for GAPDH were addedto PCR tubes at the end of the 10th cycle [27].

The levels of ING1 mRNA were estimated by scanning densitometry to beapproximately ten fold higher in senescent fibroblasts compared to youngfibroblasts. In order to see if increased mRNA levels resulted inincreased protein levels, Western blotting experiments were performedwith a rabbit polyclonal antibody that was raised against a bacteriallyexpressed glutathione-S-transferase (GST) -p33^(ING1) fusion protein andthat reacted with a 33 kDa protein in human and mouse cell Iysates.

Hs68 and NMuMG cells were harvested and 20 μg of total protein was usedin each lane. Proteins were separated by electrophoresis in 12.5%polyacrylamide/SDS gels, and transferred to membranes for 1 hour usingan electroblotter. The membranes were blocked in TBS(100 mM Tris, 150 mMNaCl) containing 10% nonfat dried milk and 0.1% Tween-20 for 2 hours.Incubation of the membranes with p33^(ING1) antiserum was performed inTBS containing 5% nonfat milk and 0.1% Tween-20 for 1 hour and thenmembranes were washed with TBST solution for 30 minutes. Horseradishperoxidase-conjugated goat anti-rabbit antibody was then applied to thefilters for 1 hour in TBST. Peroxidase activity was detected using ECL(Amersham Canada, Oakville, Ontario, Canada) and relative bandintensities were determined by scanning densitometry.

As shown in FIG. 8B, the level of p33^(ING1) protein increasesapproximately 8 fold when cells approach the end of their in vitroreplicative lifespan, consistent with results obtained using RT-PCR.

Since ING1 appears to arrest cells in Gi when overexpressed andsenescent cells are primarily arrested in the GI phase of the cell cycle[28], the level of p33^(ING1) protein was tested during the cell cycle.Quiescent, proliferation-competent NMuMG cells were serum stimulated,lysates were prepared at different times after serum addition, andsamples were analyzed by Western blotting with anti-p33 antibodies bythe method set forth above. The level of p33^(ING1) was found todecrease as cells exited from G0, to increase during late G1 and toreach a maximum in S phase. This was followed by a decrease in G2 of thecell cycle (FIG. 9B). CDK2 expression was used as a control for cellcycle progression and changed as reported previously (FIG. 9A) [29].FIG. 9c shows the results of DNA content analysis byfluorescence-activated cell sorting (FACS) in parallel culturesindicating that cells enter S phase at 16 hours under these experimentalconditions. These results indicate that ING1 is regulated followingmitogen addition to quiescent cells, with expression reaching a peakduring DNA synthesis.

To determine the effects of reducing the levels of ING1 mRNA on thereplicative lifespan of HDFs, cells were infected with a PLNCX-αS (theconstruct carrying a 182 bp fragment in the antisense orientation andrepresenting nucleotides 942 to 1,124 of the ING1 cDNA (FIG. 3)). Thisantisense fragment effectively inhibits translation of ING1 mRNA asshown previously where chronic expression of the antisense constructresulted in 90% inhibition of the expression of the endogenousp33^(ING1) protein in cells.

Amphotropic and ecotropic packaging cells that were used for infectionare capable of producing retroviruses with titers higher than 10⁶ per mlupon transient transfection which allows delivery of the retroviralconstruct to HDFs with efficiencies of approximately 90% as monitored byexpression from a retroviral-β-galactosidase construct.

"Young" HDFs at 30 MPDs were "pre-aged" by continuous subculturing untilreaching 70 MPDs. Hs68 cells at 70 mean population doublings (MPDS) wereinfected with the retroviral vector PLNCX as a control or with pLNCX-αSand were subcultured in parallel using subculturing ratios of 1:2.Infected cells were propagated an additional 10 MPDs after which 10⁵control PLCNX and 10⁵ PLCNX-αS cells at 80 MPD were split into twelve 10cm plates and cultivated for two months, with weekly refeeding usingcomplete medium. Some of the cells infected with retrovirus alone wereobserved to divide once during this time, while cells containing theING1-αS fragment continued to grow and created visible colonies.

To confirm the effect of the antisense fragment of ING1 in cells,indirect immunofluorescence with a rabbit polyclonal antibody that wasraised against p33^(ING1) was performed. Senescent vector-infectedfibroblasts and fibroblasts from colonies resulting from ING1-αSretrovirus infection were grown on glass coverslips for 48 hours at 37°C. to 60% confluence. Then the cells were fixed in 3.7% formaldehyde,washed in 0.5% Triton X100 and in 0.05% Tween 20 in PBS solution for 10minutes each at room temperature. The cells were incubated with a 1:100dilution of rabbit p33^(ING1) antiserum for 30 min, washed in PBS with0.05% Tween, incubated with goat anti-rabbit IgG-biotin antibody andthen with streptavidin conjugated Texas Red. Samples were examined witha Zeiss Axiophot fluorescence microscope and images were photographed onKodak TMAX 400 film.

Staining with anti-p33 antibody was observed in the nuclear compartmentof senescent cells containing control virus but not in cells obtainedfrom colonies that had received antisense p33 retrovirus. These resultscorroborate the previous observations that p33^(ING1) is a nuclearprotein and confirmed that the levels of p33^(ING1) protein decrease incells from colonies resulting from ING1-αS retrovirus infection. Similarresults were seen in cells from 3 individual colonies and from 20independent senescent cells containing control retrovirus.

To estimate the efficiency with which down regulation of the ING1 geneby infection with PLCNX-αS was able to extend the proliferative lifespanof normal fibroblasts, the number of cells in each colony was counted.Results of these calculations are shown in FIG. 10 in which colonieswere divided into 4 groups depending upon the number of cells in thecolony. Most colonies contained 100-159 cells, therefore if cellsdivided in an arithmetic progression (2,4,8 . . . n) this classcorresponds to approximately 7 additional MPDs (2⁷ =128). Colonies inthe largest category (220-280) correspond to 8 cell doublings (2⁸ =256).Similar results were obtained in two separate trials and stronglyindicate that down regulation of p33^(ING1) protein is sufficient toextend the proliferative lifespan of normal fibroblasts by approximately10%, as previously reported for the p53 tumor suppressor gene [30].

Modification of the above-described modes of carrying out variousembodiments of this invention will be apparent to those skilled in theart following the teachings of this invention as set forth herein. Theexamples described above are not limiting, but are merely exemplary ofthis invention, the scope of which is defined by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 23                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1902 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 109..741                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - CTGACCCGAG GGTGGGGCCG CGCGTGGCCG TGGAAACAGA TCCTGAAGGA GC - #TAGACGAG         60                                                                          #CTG CAC     117TCAGTCG CGAGACAGAC GGGGCGCAGA AGCGGCGG ATG                    #                 Met - # Leu His                                             # 1                                                                           - TGT GTG CAG CGC GCG CTG ATC CGC AGC CAG GA - #G CTG GGC GAC GAG AAG          165                                                                          Cys Val Gln Arg Ala Leu Ile Arg Ser Gln Gl - #u Leu Gly Asp Glu Lys           #      15                                                                     - ATC CAG ATC GTG AGC CAG ATG GTG GAG CTG GT - #G GAG AAC CGC ACG CGG          213                                                                          Ile Gln Ile Val Ser Gln Met Val Glu Leu Va - #l Glu Asn Arg Thr Arg           # 35                                                                          - CAG GTG GAC AGC CAC GTG GAG CTG TTC GAG GC - #G CAG CAG GAG CTG GGC          261                                                                          Gln Val Asp Ser His Val Glu Leu Phe Glu Al - #a Gln Gln Glu Leu Gly           #                 50                                                          - GAC ACA GTG GGC AAC AGC GGC AAG GTT GGC GC - #G GAC AGG CCC AAT GGC          309                                                                          Asp Thr Val Gly Asn Ser Gly Lys Val Gly Al - #a Asp Arg Pro Asn Gly           #             65                                                              - GAT GCG GTA GCG CAG TCT GAC AAG CCC AAC AG - #C AAG CGC TCA CGG CGG          357                                                                          Asp Ala Val Ala Gln Ser Asp Lys Pro Asn Se - #r Lys Arg Ser Arg Arg           #         80                                                                  - CAG CGC AAC AAC GAG AAC CGT GAG AAC GCG TC - #C AGC AAC CAC GAC CAC          405                                                                          Gln Arg Asn Asn Glu Asn Arg Glu Asn Ala Se - #r Ser Asn His Asp His           #     95                                                                      - GAC GAC GGC GCC TCG GGC ACA CCC AAG GAG AA - #G AAG GCC AAG ACC TCC          453                                                                          Asp Asp Gly Ala Ser Gly Thr Pro Lys Glu Ly - #s Lys Ala Lys Thr Ser           100                 1 - #05                 1 - #10                 1 -       #15                                                                           - AAG AAG AAG AAG CGC TCC AAG GCC AAG GCG GA - #G CGA GAG GCG TCC CCT          501                                                                          Lys Lys Lys Lys Arg Ser Lys Ala Lys Ala Gl - #u Arg Glu Ala Ser Pro           #               130                                                           - GCC GAC CTC CCC ATC GAC CCC AAC GAA CCC AC - #G TAC TGT CTG TGC AAC          549                                                                          Ala Asp Leu Pro Ile Asp Pro Asn Glu Pro Th - #r Tyr Cys Leu Cys Asn           #           145                                                               - CAG GTC TCC TAT GGG GAG ATG ATC GGC TGC GA - #C AAC GAC GAG TGC CCC          597                                                                          Gln Val Ser Tyr Gly Glu Met Ile Gly Cys As - #p Asn Asp Glu Cys Pro           #       160                                                                   - ATC GAG TGG TTC CAC TTC TCG TGC GTG GGG CT - #C AAT CAT AAA CCC AAG          645                                                                          Ile Glu Trp Phe His Phe Ser Cys Val Gly Le - #u Asn His Lys Pro Lys           #   175                                                                       - GGC AAG TGG TAC TGT CCC AAG TGC CGG GGG GA - #G AAC GAG AAG ACC ATG          693                                                                          Gly Lys Trp Tyr Cys Pro Lys Cys Arg Gly Gl - #u Asn Glu Lys Thr Met           180                 1 - #85                 1 - #90                 1 -       #95                                                                           - GAC AAA GCC CTG GAG AAA TCC AAA AAA GAG AG - #G GCT TAC AAC AGG TAG          741                                                                          Asp Lys Ala Leu Glu Lys Ser Lys Lys Glu Ar - #g Ala Tyr Asn Arg  *            #               210                                                           - TTTGTGGACA GGCGCCTGGT GTGAGGAGGA CAAAATAAAC CGTGTATTTA TT - #ACATTGCT        801                                                                          - GCCTTTGTTG AGGTGCAAGG AGTGTAAAAT GTATATTTTT AAAGAATGTT AG - #AAAAGGAA        861                                                                          - CCATTCCTTT CATAGGGATG GCAGTGATTC TGTTTGCCTT TTGTTTTCAT TG - #GTACACGT        921                                                                          - GTAACAAGAA AGTGGTCTGT GGATCAGCAT TTTAGAAACT ACAAATATAG GT - #TTGATTCA        981                                                                          - ACACTTAAGT CTCAGACTGA TTTCTTGCGG GAGGAGGGGG ACTAAACTCA CC - #CTAACACA       1041                                                                          - TTAAATGTGG AAGGAAAATA TTTCATTAGC TTTTTTATTT TAATACAAGT AA - #TATTATTA       1101                                                                          - CTTTATGAAC AATTTTTTTT AATTGGCCAT GTCGCCAAAA ATACAGCCTA TA - #GTAAATGT       1161                                                                          - GTTTCTTGCT GCCATGATGT ATATCCATAT AACAATTCAG TAACAAAGGT TT - #AAAGTTTG       1221                                                                          - AAGATTATTT TTTAAAAAGG TAAAAGGTTA AATTTTACAT GACAGATATT TT - #ATCTATTG       1281                                                                          - GCCTGTTCCC CAAATGGCCA TTTTAAAATG CTTGGGTACA CTTCTCTTAA GT - #GGTCTAGT       1341                                                                          - CAAGGAACCT CAAGTCATGC TTTTGCTATC ACCAATCATA GTGTACCCAT CT - #TTAATTTA       1401                                                                          - TATCAGGTGT ATAAATGTAC ATTTCCAAAT GAACTTGCAC TGTAATATTA TA - #ATTGGAAG       1461                                                                          - TGCAGTCAGC AGTAGCTGTC GGAGCTAATG TCACAATTAT GTGCAAAGGT GT - #GCTTCCTG       1521                                                                          - CTGTATGTGA GCTGTAAAAA TGTTACGTGA AGAAATAAAT GAAACTTGGC CA - #GTTTGTTC       1581                                                                          - CTCTAGTAGT ATATTTAATT TTGACATAAG TAACTTTTAA AATTTGTCTT AA - #AAATTTAT       1641                                                                          - ACACCAGCAA TTTAGACAAA GCCTTAAGCA AATTTTGTAT TATTGTTCTC AC - #TTATTATT       1701                                                                          - AATAATGAAG TAGAAGTTAC TTAATTGCCA GCAAATAAAT ACGTGTCAAA AA - #AGAATCTG       1761                                                                          - TATTCAGACC CCTGGGGTCA GGAAATTACT GCCCCACTTG TCAAGTTCAG CC - #CACCATCT       1821                                                                          - GTTTGAACAT TATATGAAGT TTAAATTCTA GTGTCCATAA ATAAAGTTTC AG - #CGGCACCC       1881                                                                          #                1902AA A                                                     - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 210 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Leu His Cys Val Gln Arg Ala Leu Ile Ar - #g Ser Gln Glu Leu Gly         #                 15                                                          - Asp Glu Lys Ile Gln Ile Val Ser Gln Met Va - #l Glu Leu Val Glu Asn         #             30                                                              - Arg Thr Arg Gln Val Asp Ser His Val Glu Le - #u Phe Glu Ala Gln Gln         #         45                                                                  - Glu Leu Gly Asp Thr Val Gly Asn Ser Gly Ly - #s Val Gly Ala Asp Arg         #     60                                                                      - Pro Asn Gly Asp Ala Val Ala Gln Ser Asp Ly - #s Pro Asn Ser Lys Arg         # 80                                                                          - Ser Arg Arg Gln Arg Asn Asn Glu Asn Arg Gl - #u Asn Ala Ser Ser Asn         #                 95                                                          - His Asp His Asp Asp Gly Ala Ser Gly Thr Pr - #o Lys Glu Lys Lys Ala         #           110                                                               - Lys Thr Ser Lys Lys Lys Lys Arg Ser Lys Al - #a Lys Ala Glu Arg Glu         #       125                                                                   - Ala Ser Pro Ala Asp Leu Pro Ile Asp Pro As - #n Glu Pro Thr Tyr Cys         #   140                                                                       - Leu Cys Asn Gln Val Ser Tyr Gly Glu Met Il - #e Gly Cys Asp Asn Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Cys Pro Ile Glu Trp Phe His Phe Ser Cy - #s Val Gly Leu Asn His         #               175                                                           - Lys Pro Lys Gly Lys Trp Tyr Cys Pro Lys Cy - #s Arg Gly Glu Asn Glu         #           190                                                               - Lys Thr Met Asp Lys Ala Leu Glu Lys Ser Ly - #s Lys Glu Arg Ala Tyr         #       205                                                                   - Asn Arg                                                                         210                                                                       - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #                24TCAC GACA                                                  - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #              26  TAGA GCCAGG                                                - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #                24ACGG CAGT                                                  - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #              26  CAAC GTCGAG                                                - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #              26  TCGA TGGATG                                                - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #        34        CTAG CTTGCCAAAC CTAC                                       - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 2061 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 16..900                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #AGA ATT CCC AGA TAT      51TG TGC ACG GCG ACG                                #Pro Leu Cys Thr Ala Thr Arg Ile Pro Arg T - #yr                              #                10                                                           - AGC AGT AGC AGT GAT CCC GGG CCT GTG GCT CG - #G GGC CGG GGC TGC AGT           99                                                                          Ser Ser Ser Ser Asp Pro Gly Pro Val Ala Ar - #g Gly Arg Gly Cys Ser           #         25                                                                  - TCG GAC CGC CTC CCG CGA CCC GCG GGG CCG GC - #T CGG AGA CAG TTT CAG          147                                                                          Ser Asp Arg Leu Pro Arg Pro Ala Gly Pro Al - #a Arg Arg Gln Phe Gln           #     40                                                                      - GCC GCA TCT TTG CTG ACC CGA GGG TGG GGC CG - #C GCG TGG CCG TGG AAA          195                                                                          Ala Ala Ser Leu Leu Thr Arg Gly Trp Gly Ar - #g Ala Trp Pro Trp Lys           # 60                                                                          - CAG ATC CTG AAG GAG CTA GAC GAG TGC TAC GA - #G CGC TTC AGT CGC GAG          243                                                                          Gln Ile Leu Lys Glu Leu Asp Glu Cys Tyr Gl - #u Arg Phe Ser Arg Glu           #                 75                                                          - ACA GAC GGG GCG CAG AAG CGG CGG ATG CTG CA - #C TGT GTG CAG CGC GCG          291                                                                          Thr Asp Gly Ala Gln Lys Arg Arg Met Leu Hi - #s Cys Val Gln Arg Ala           #             90                                                              - CTG ATC CGC AGC CAG GAG CTG GGC GAC GAG AA - #G ATC CAG ATC GTG AGC          339                                                                          Leu Ile Arg Ser Gln Glu Leu Gly Asp Glu Ly - #s Ile Gln Ile Val Ser           #        105                                                                  - CAG ATG GTG GAG CTG GTG GAG AAC CGC ACG CG - #G CAG GTG GAC AGC CAC          387                                                                          Gln Met Val Glu Leu Val Glu Asn Arg Thr Ar - #g Gln Val Asp Ser His           #   120                                                                       - GTG GAG CTG TTC GAG GCG CAG CAG GAG CTG GG - #C GAC ACA GTG GGC AAC          435                                                                          Val Glu Leu Phe Glu Ala Gln Gln Glu Leu Gl - #y Asp Thr Val Gly Asn           125                 1 - #30                 1 - #35                 1 -       #40                                                                           - AGC GGC AAG GTT GGC GCG GAC AGG CCC AAT GG - #C GAT GCG GTA GCG CAG          483                                                                          Ser Gly Lys Val Gly Ala Asp Arg Pro Asn Gl - #y Asp Ala Val Ala Gln           #               155                                                           - TCT GAC AAG CCC AAC AGC AAG CGC TCA CGG CG - #G CAG CGC AAC AAC GAG          531                                                                          Ser Asp Lys Pro Asn Ser Lys Arg Ser Arg Ar - #g Gln Arg Asn Asn Glu           #           170                                                               - AAC CGT GAG AAC GCG TCC AGC AAC CAC GAC CA - #C GAC GAC GGC GCC TCG          579                                                                          Asn Arg Glu Asn Ala Ser Ser Asn His Asp Hi - #s Asp Asp Gly Ala Ser           #       185                                                                   - GGC ACA CCC AAG GAG AAG AAG GCC AAG ACC TC - #C AAG AAG AAG AAG CGC          627                                                                          Gly Thr Pro Lys Glu Lys Lys Ala Lys Thr Se - #r Lys Lys Lys Lys Arg           #   200                                                                       - TCC AAG GCC AAG GCG GAG CGA GAG GCG TCC CC - #T GCC GAC CTC CCC ATC          675                                                                          Ser Lys Ala Lys Ala Glu Arg Glu Ala Ser Pr - #o Ala Asp Leu Pro Ile           205                 2 - #10                 2 - #15                 2 -       #20                                                                           - GAC CCC AAC GAA CCC ACG TAC TGT CTG TGC AA - #C CAG GTC TCC TAT GGG          723                                                                          Asp Pro Asn Glu Pro Thr Tyr Cys Leu Cys As - #n Gln Val Ser Tyr Gly           #               235                                                           - GAG ATG ATC GGC TGC GAC AAC GAC GAG TGC CC - #C ATC GAG TGG TTC CAC          771                                                                          Glu Met Ile Gly Cys Asp Asn Asp Glu Cys Pr - #o Ile Glu Trp Phe His           #           250                                                               - TTC TCG TGC GTG GGG CTC AAT CAT AAA CCC AA - #G GGC AAG TGG TAC TGT          819                                                                          Phe Ser Cys Val Gly Leu Asn His Lys Pro Ly - #s Gly Lys Trp Tyr Cys           #       265                                                                   - CCC AAG TGC CGG GGG GAG AAC GAG AAG ACC AT - #G GAC AAA GCC CTG GAG          867                                                                          Pro Lys Cys Arg Gly Glu Asn Glu Lys Thr Me - #t Asp Lys Ala Leu Glu           #   280                                                                       - AAA TCC AAA AAA GAG AGG GCT TAC AAC AGG TA - #G TTTGTGGACA GGCGCCTGGT        920                                                                          #*s Ser Lys Lys Glu Arg Ala Tyr Asn Arg                                       285                 2 - #90                 2 - #95                           - GTGAGGAGGA CAAAATAAAC CGTGTATTTA TTACATTGCT GCCTTTGTTG AG - #GTGCAAGG        980                                                                          - AGTGTAAAAT GTATATTTTT AAAGAATGTT AGAAAAGGAA CCATTCCTTT CA - #TAGGGATG       1040                                                                          - GCAGTGATTC TGTTTGCCTT TTGTTTTCAT TGGTACACGT GTAACAAGAA AG - #TGGTCTGT       1100                                                                          - GGATCAGCAT TTTAGAAACT ACAAATATAG GTTTGATTCA ACACTTAAGT CT - #CAGACTGA       1160                                                                          - TTTCTTGCGG GAGGAGGGGG ACTAAACTCA CCCTAACACA TTAAATGTGG AA - #GGAAAATA       1220                                                                          - TTTCATTAGC TTTTTTATTT TAATACAAGT AATATTATTA CTTTATGAAC AA - #TTTTTTTT       1280                                                                          - AATTGGCCAT GTCGCCAAAA ATACAGCCTA TAGTAAATGT GTTTCTTGCT GC - #CATGATGT       1340                                                                          - ATATCCATAT AACAATTCAG TAACAAAGGT TTAAAGTTTG AAGATTATTT TT - #TAAAAAGG       1400                                                                          - TAAAAGGTTA AATTTTACAT GACAGATATT TTATCTATTG GCCTGTTCCC CA - #AATGGCCA       1460                                                                          - TTTTAAAATG CTTGGGTACA CTTCTCTTAA GTGGTCTAGT CAAGGAACCT CA - #AGTCATGC       1520                                                                          - TTTTGCTATC ACCAATCATA GTGTACCCAT CTTTAATTTA TATCAGGTGT AT - #AAATGTAC       1580                                                                          - ATTTCCAAAT GAACTTGCAC TGTAATATTA TAATTGGAAG TGCAGTCAGC AG - #TAGCTGTC       1640                                                                          - GGAGCTAATG TCACAATTAT GTGCAAAGGT GTGCTTCCTG CTGTATGTGA GC - #TGTAAAAA       1700                                                                          - TGTTACGTGA AGAAATAAAT GAAACTTGGC CAGTTTGTTC CTCTAGTAGT AT - #ATTTAATT       1760                                                                          - TTGACATAAG TAACTTTTAA AATTTGTCTT AAAAATTTAT ACACCAGCAA TT - #TAGACAAA       1820                                                                          - GCCTTAAGCA AATTTTGTAT TATTGTTCTC ACTTATTATT AATAATGAAG TA - #GAAGTTAC       1880                                                                          - TTAATTGCCA GCAAATAAAT ACGTGTCAAA AAAGAATCTG TATTCAGACC CC - #TGGGGTCA       1940                                                                          - GGAAATTACT GCCCCACTTG TCAAGTTCAG CCCACCATCT GTTTGAACAT TA - #TATGAAGT       2000                                                                          - TTAAATTCTA GTGTCCATAA ATAAAGTTTC AGCGGCACCC CAAAAAAAAA AA - #AAAAAAAA       2060                                                                          #             2061                                                            - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 294 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Met Pro Leu Cys Thr Ala Thr Arg Ile Pro Ar - #g Tyr Ser Ser Ser Ser         #                 15                                                          - Asp Pro Gly Pro Val Ala Arg Gly Arg Gly Cy - #s Ser Ser Asp Arg Leu         #             30                                                              - Pro Arg Pro Ala Gly Pro Ala Arg Arg Gln Ph - #e Gln Ala Ala Ser Leu         #         45                                                                  - Leu Thr Arg Gly Trp Gly Arg Ala Trp Pro Tr - #p Lys Gln Ile Leu Lys         #     60                                                                      - Glu Leu Asp Glu Cys Tyr Glu Arg Phe Ser Ar - #g Glu Thr Asp Gly Ala         # 80                                                                          - Gln Lys Arg Arg Met Leu His Cys Val Gln Ar - #g Ala Leu Ile Arg Ser         #                 95                                                          - Gln Glu Leu Gly Asp Glu Lys Ile Gln Ile Va - #l Ser Gln Met Val Glu         #           110                                                               - Leu Val Glu Asn Arg Thr Arg Gln Val Asp Se - #r His Val Glu Leu Phe         #       125                                                                   - Glu Ala Gln Gln Glu Leu Gly Asp Thr Val Gl - #y Asn Ser Gly Lys Val         #   140                                                                       - Gly Ala Asp Arg Pro Asn Gly Asp Ala Val Al - #a Gln Ser Asp Lys Pro         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Ser Lys Arg Ser Arg Arg Gln Arg Asn As - #n Glu Asn Arg Glu Asn         #               175                                                           - Ala Ser Ser Asn His Asp His Asp Asp Gly Al - #a Ser Gly Thr Pro Lys         #           190                                                               - Glu Lys Lys Ala Lys Thr Ser Lys Lys Lys Ly - #s Arg Ser Lys Ala Lys         #       205                                                                   - Ala Glu Arg Glu Ala Ser Pro Ala Asp Leu Pr - #o Ile Asp Pro Asn Glu         #   220                                                                       - Pro Thr Tyr Cys Leu Cys Asn Gln Val Ser Ty - #r Gly Glu Met Ile Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Cys Asp Asn Asp Glu Cys Pro Ile Glu Trp Ph - #e His Phe Ser Cys Val         #               255                                                           - Gly Leu Asn His Lys Pro Lys Gly Lys Trp Ty - #r Cys Pro Lys Cys Arg         #           270                                                               - Gly Glu Asn Glu Lys Thr Met Asp Lys Ala Le - #u Glu Lys Ser Lys Lys         #       285                                                                   - Glu Arg Ala Tyr Asn Arg                                                         290                                                                       - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 44               GAAC GCGTAATACG CCTCACTATA GGGA                            - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #  18              CC                                                         - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                # 20               CTTC                                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #                23GAAC GCG                                                   - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #21                AGGG A                                                     - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                # 20               ACCA                                                       - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                # 20               GCTT                                                       - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                # 20               TCTC                                                       - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                # 20               CACT                                                       - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #                24GGTC GTAT                                                  - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #                24GTGA AGAC                                                  - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                # 20               ATGG                                                       - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #                23GATT AAA                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated and purified nucleic acid sequencewhich encodes the amino acid sequence of SEQ ID NO:
 10. 2. A recombinantexpression vector comprising a nucleic acid sequence which encodes theamino acid sequence of SEQ ID NO:
 10. 3. A cell in culture transformedwith the recombinant expression vector of claim 2.